Polyamine biosynthetic enzymes as drug targets in parasitic protozoa

Molecular, biochemical and genetic characterization of ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase establishes that these polyamine-biosynthetic enzymes are essential for growth and survival of the agents that cause African sleeping sickness, Chagas' disease, leishmaniasis and malaria. These enzymes exhibit features that differ significantly between the parasites and the human host. Therefore it is conceivable that exploitation of such differences can lead to the design of new inhibitors that will selectively kill the parasites while exerting minimal, or at least tolerable, effects on the parasite-infected patient.     

Application of rbcL based molecular diversity analysis to algae in wastewater treatment plants

The molecular diversity of algae in the final clarifier or denitrification filter outfall from three wastewater treatment plants (WWTPs) with activated sludge based treatment was analyzed using the rbcL gene as a phylogenetic marker. The rbcL gene encodes the large subunit of the CO(2) fixing enzyme RuBisCO. Among algae identified at the WWTPs were diatoms, green algae, cyanobacteria, Eustigmatophyceae, and unknown heterokonts. A high level of diversity was observed within WWTPs with 19-24 unique rbcL sequences detected at each plant. Algae composition also varied between treatment plants. Our results show that the rbcL gene can be used as a phylogenetic marker for algae diversity analysis in a wastewater treatment context.     

Polyamine titer and biosynthetic enzymes during tuber formation ofHelianthus tuberosus

During the formation ofHelianthus tuberosus tubers the activities of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC), examined in medullary parenchyma cells, increase with the increase in weight of the tuber. The ornithine decarboxylase (ODC) activity is about 100-fold less with respect to ADC activity, and it was detected only during the deceleration phase of the growth curve. Spermidine and spermine content are strictly related to the SAMDC activity and tuber growth. The increase of ADC and SAMDC activity is directly related to cell extension and increase in weight. The limited area of cell division in parenchyma tissue found during the first stage of tuber formation could justify the low ODC activity. The data suggest that ADC affects mainly growth processes, while ODC seems to be preferentially related to cell division.     

Serpins in plants and green algae

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.     

Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

Chloroplast evolution in algae and land plants

Eukaryotes contain a chimeric assembly of genomes, each localized in a specialized subcellular compartment. The successful survival of an organism requires that these sequestered genomes be viewed as dependent variables in a coevolutionary complex. This discussion focuses on chloroplast evolution. A selected review of information available on chloroplast diversity is presented, followed by an analysis of the genetic modifications which may have occurred in the conversion of a free-living ancestral photosynthetic prokaryote into an organelle that has an obligately dependent and highly efficient interplay with the nuclear genome.     

Ascorbate as a biosynthetic precursor in plants

BACKGROUND AND AIMS: l-Ascorbate (vitamin C) has well-documented roles in many aspects of redox control and anti-oxidant activity in plant cells. This Botanical Briefing highlights recent developments in another aspect of l-ascorbate metabolism: its function as a precursor for specific processes in the biosynthesis of organic acids. SCOPE: The Briefing provides a summary of recent advances in our understanding of l-ascorbate metabolism, covering biosynthesis, translocation and functional aspects. The role of l-ascorbate as a biosynthetic precursor in the formation of oxalic acid, l-threonic acid and l-tartaric acid is described, and progress in elaborating the mechanisms of the formation of these acids is reviewed. The potential conflict between the two roles of l-ascorbate in plant cells, functional and biosynthetic, is highlighted. CONCLUSIONS: Recent advances in the understanding of l-ascorbate catabolism and the formation of oxalic and l-tartaric acids provide compelling evidence for a major role of l-ascorbate in plant metabolism. Combined experimental approaches, using classic biochemical and emerging 'omics' technologies, have provided recent insight to previously under-investigated areas.     

Chlorophylla biosynthetic routes and chlorophylla chemical heterogeneity in plants

A six-branched chlorophyll a biosynthetic pathway instead of a four-branched pathway has been proposed as being responsible for the formation of chlorophyll a in green plants. The several biosynthetic routes that make up the pathway have been described as leading to the formation of ten chemically different groups of chlorophyll a species. The latter differ from one another by one or more of the following modifications: (a) by having a vinyl or ethyl group at position 4 of the macrocycle, (b) by the nature of the long-chain fatty alcohols at position 7 of the macrocycle, and (c) by having a 6-membered lactone ring instead of a 5-membered cyclopentanone ring. The chemical structure of several of the metabolic intermediates of that pathway and of some of the chlorophyll a species have now been determined by primary chemical derivatization methods coupled to spectrofluorometric, nuclear magnetic resonance and mass spectral analyses. The formation of highly organized photosynthetic membranes in which some of the chlorophyll alpha molecules are specifically oriented is ascribed to the multiplicity of chlorophyll biosynthetic routes which result in the formation of multiple chlorophyll alpha chemical species. Proper orientation of chlorophyll in the photosynthetic membranes is visualized as being controlled by peripheral group modifications that either modulate the polarity of the Chl chromophore or control the magnitude of the net positive charge on the central Mg atom. Finally it is proposed that in addition to the proper orientation of chlorophyll a, chemical heterogeneity of the chlorophyll chromophores in the photosynthetic reaction centers is mandatory for efficient charge separation, and proper vectorial electron transfer.     

Exploring biosynthetic diversity with trichodiene synthase

Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP(i)). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.85A resolution X-ray crystal structure of the complex with Mg(2+) (3)-PP(i) and the benzyl triethylammonium cation, which is a bulkier mimic of the bisabolyl carbocation intermediate in catalysis. Taken together, these findings show that the active site of trichodiene synthase is sufficiently flexible to accommodate bulkier and electronically-diverse substrates and intermediates, which could indicate additional potential for the biosynthetic utility of this terpenoid cyclase.     

Polyamine research in plants – a changing perspective

Our understanding of the precise role(s) of polyamines (PAs) in various plant developmental and morphogenetic processes has advanced considerably by the ability to manipulate PA biosynthetic pathways using polyamine biosynthesis inhibitors, PA- mutants and by adopting various transgenic strategies. The cDNA for almost every biosynthesis pathway enzyme has been isolated and cloned in a number of systems. This review briefly summarizes our current understanding of the genetic control of PA metabolism in different model plant systems.     

Manipulation of hormone biosynthetic genes in transgenic plants

Modification of plant hormone biosynthesis through the introduction of bacterial genes is a natural form of genetic engineering, which has been exploited in numerous studies on hormone function. Recently, biosynthetic pathways have been largely elucidated for most of the plant hormone classes, and genes encoding many of the enzymes have been cloned. These advances offer new opportunities to manipulate hormone content in order to study their mode of action and the regulation of their biosynthesis. Furthermore, this technology is providing the means to introduce agriculturally useful traits into crops.     

Genomic diversity and biosynthetic capabilities of sponge-associated chlamydiae

Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems.Subject terms: Symbiosis, Phylogenetics, Metagenomics, Comparative genomics, Microbiome     

Polyamine catabolism in higher plants: Characterization of pyrroline dehydrogenase

Both mono-and dicotyledonous species catabolize putrescine to -aminobutyric acid (GABA), but by two different pathways. GABA is the major labeled product in pea shoots and oat leaves fed with a 2–4 h pulse of [1,4-¹⁴C]-putrescine (Put) or [1,4-tetramethylene-¹⁴C]-spermidine (Spd), respectively. In the presence of 1–10 M gabaculine, a specific inhibitor of GABA: pyruvate-transaminase, the label appearing in GABA increases 2 to 7-fold, which indicates that the transamination reaction is a major fate of GABA formed from Put or Spd in vivo. The conversions to GABA were demonstrated in vitro in coupled assays involving diamine oxidase from pea or polyamine oxidase from oat, and pyrroline dehydrogenase (PYRR-DH). The latter enzyme from either pea or oat is strictly NAD-dependent and is specific for pyrroline. The optimal temperature (40–45°C) and pH (7.5–8.0) are similar to those of bacterial PYRR-DH. In all cases the enzyme was inhibited by the NAD analogs thionicotinamide and aminopyridine dinucleotide (0.1–1.0 mM). In addition to pea and oat, PYRR-DH was also detected in corn, barley, soybean and broadbean. Di- and polyamine oxidase are released by enzymes which degrade the cell wall, while PYRR-DH remains associated with the protoplast.